Use of Acacia Gum to isolate and preserve the biological receptor on a biosensor

ABSTRACT

Compositions and methods for the reversible preservation of biological samples are provided. The compositions include  Acacia  Gum, including derivations and modifications thereof which are useful as a reversible preservation solution. A method is provided for using  Acacia  Gum to isolate and reversibly preserve a biological specimen in a dormant state at room temperature for an extended period with minimal damage to the specimen. The compositions and methods disclosed may also be used to create reversibly preserved biological specimens and biological receptors for use in biosensors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/000,727, which is incorporated herein in its entirety by reference,and which claims the benefit and priority of U.S. ProvisionalApplication No. 60/250,798, filed Dec. 1, 2000, entitled “Method ofProtection of Biosensor Surface,” and U.S. Provisional Application No.60/250,799, filed Dec. 1, 2000, entitled “Method for Protection ofBiological Material,” both of which are also incorporated herein intheir entirety by reference.

TECHNICAL FIELD

The present invention relates generally to the field of biologicalsample preservation and, more particularly, to a method of using asolution of Acacia Gum to preserve a biological specimen in a dormantstate and, later, using an aqueous solution to restore the specimenunharmed to its isolated condition.

BACKGROUND OF THE INVENTION

Various methods for the preservation of biological specimens haveevolved over the years. Modern specimen preparation techniques formicrobiology and electron microscopy typically include dehydration andimmobilization, both of which are irreversible and often damage theintegrity of the specimen.

Dehydration using chemicals or freezing temperatures typically causesstructural damage to biological tissues. Chemicals may destroy theoverall quality of the specimen, including the particularcharacteristics of interest to the scientist. Rapid freeze-drying oftenproduces crystalline structures that are destructive to most biologicaltissues. The result of dehydration is a biological sample that has beensignificantly altered, beyond repair, from its natural state.

Immobilization of a biological sample within a polymer typicallyinvolves curing, using elevated temperatures or ultraviolet radiation,both of which are detrimental to specimen quality. The polymers andresins typically used for sample preparation today form a hard plasticwhen cured. Once a sample has been cured, the biological material cannotbe restored to its isolated state.

Biological specimen preservation techniques are of particular concern inthe preparation of biosensors. Biosensors are used in the health andenvironmental sciences for rapid detection of specific substances.Biosensors are currently used to detect the presence of pesticides,herbicides, and other compounds; to detect the presence of organiccompounds such as alcohols, ammonia, and metals; and, to detect thepresence of specific bacteria including algae, fungi, and pathogenicorganisms such as Escherichia coli (E. coli) and Salmonella. Potentialapplications for biosensors include sensing pollution and microbialcontamination of air and water, clinical diagnosis of medicalconditions, fermentation analysis and control, monitoring and analysisof industrial gases and liquids, monitoring of mining conditions andsensing toxic gases.

Biosensors often have a very short shelf life because the antibody orother biological receptor degrades rapidly when exposed to theenvironment. Like other biological samples, biological receptors needisolation and protection from the environment until ready for use. Infield applications, especially, a variety of biological receptors may beneeded at any time, depending upon the conditions.

There is an unsatisfied need in the art for biological samples that canbe protected and preserved without altering or destroying the biologicaltissue. The demand for safe transport and prolonged storage ofbiological samples today requires preservation techniques that maintainthe integrity and quality of the biological sample. Sensitive biologicalreceptors used in biosensors need to be isolated from the environment,without damaging the receptor, until ready for use. None of the specimenpreparation techniques in the art currently meet these needs.

There is also a need in the art for biological samples that can berestored to their isolated or prepared state after immobilization, withminimal damage, for later study or use. The current techniques ofdehydration and immobilization are irreversible and destroy sampleviability. Restoration is particularly critical for the biologicalreceptors in biosensors, which are especially sensitive. There is aneed, therefore, for a preservation technique that is both harmless andreversible.

SUMMARY OF THE INVENTION

The above and other needs are met by the present invention which, statedgenerally, provides a method of using Acacia Gum to isolate and preservebiological material without damage to the specimen. The presentinvention further provides reversible techniques for using Acacia Gumthat maintain the integrity and viability of biological specimens, evenafter prolonged storage at room temperature.

In one aspect of the invention, a reversibly preserved biologicalspecimen is provided. The specimen in an isolated condition has beencombined with an effective amount of a solution of solid Acacia Gumdissolved in water. The suspension has been cured in ambient conditionsto form a solid that can later be restored to a suspension. In oneaspect, the suspension is capable of being separated so that thebiological specimen can be restored to its former, isolated condition.In one embodiment, the biological specimen may include a separatecontainer holding an effective amount of aqueous solution to restore thesuspension by irrigating the solid in ambient conditions with theaqueous solution. The aqueous solution used to irrigate the solid mayinclude distilled water, a buffer of 3-(N-morpholino) propanesulfonicacid, and one or more salts such as potassium chloride, sodium chloride,magnesium chloride, and/or calcium chloride.

In another aspect of the invention, a method of reversibly preserving abiological specimen includes the steps of combining the specimen in anisolated condition with an effective amount of an Acacia Gum solution toform a suspension and, then, curing the suspension in ambient conditionsto form a solid. The preservation method may also include the steps ofirrigating the solid in ambient conditions with an effective amount ofan aqueous solution to restore the suspension and then separating thesolution from the specimen to restore the specimen to its former,isolated condition.

In one embodiment, the Acacia Gum solution is formed by dissolving solidAcacia Gum in distilled water. The combining step may include immersingthe specimen in the Acacia Gum solution. The curing step may includestirring the suspension.

In one embodiment, the aqueous solution used to irrigate the solid mayinclude distilled water, a buffer, and one or more salts such aspotassium chloride, sodium chloride, magnesium chloride, and/or calciumchloride. The buffer may be 3-(N-morpholino) propanesulfonic acid.

The biological specimens suitable for preservation may bemicroorganisms, viruses, bacteria, phages, antibodies, antigens, DNA,RNA, receptors, enzymes, proteins, biochemicals, yeast, fungi, plant andanimal cells and extracts, semen, sperm, ova, blood, tissue samples,cell samples, urine, saliva, lymphatic fluid, skin, hair, bones, or bonemarrow. In one embodiment, the biological specimen may be a biosensor.

In another aspect of the invention, a method of fabricating a reversiblypreserved biological specimen includes the steps of combining thebiological specimen in an isolated condition with an effective amount ofan Acacia Gum solution to form a suspension and, then, curing thesuspension in ambient conditions to form a solid that can later berestored to a suspension. In one aspect, the suspension is capable ofbeing separated so that the biological specimen can be restored to itsformer, isolated condition.

In one embodiment, the Acacia Gum solution used in this method offabrication is formed by dissolving solid Acacia Gum in distilled water.The curing step may include stirring the suspension. The combining stepmay include immersing the specimen.

In one embodiment, the method may include providing an effective amountof aqueous solution to restore the suspension by irrigating the solid inambient conditions with the aqueous solution. The aqueous solution usedto irrigate the solid may include distilled water, a buffer of3-(N-morpholino) propanesulfonic acid, and one or more salts such aspotassium chloride, sodium chloride, magnesium chloride, and/or calciumchloride.

In another aspect of the invention, a method of restoring the biologicalreceptor includes the steps of irrigating the solid in ambientconditions with an effective amount of an aqueous solution to restorethe suspension and, then, separating the solution from the biologicalreceptor such that the biological receptor is substantially restored toits former, isolated condition. In one embodiment, the aqueous solutionused to irrigate the solid may include distilled water, a buffer, andone or more salts such as potassium chloride, sodium chloride, magnesiumchloride, and/or calcium chloride. The buffer may be 3-(N-morpholino)propanesulfonic acid.

In another aspect of the invention, a biosensor having a reversiblypreserved biological receptor includes a signal transducer, an interfaceconnected to the signal transducer, and a solid containing thebiological receptor. The solid has been formed by curing a suspension inambient conditions. The suspension includes the biological receptor inits prepared condition and an effective amount of an Acacia Gumsolution. The suspension is capable of being separated so that thebiological receptor can be restored to its former, prepared condition.

In one embodiment, the Acacia Gum solution is formed by dissolving solidAcacia Gum in distilled water. The biological receptors suitable forpreservation may be microorganisms, viruses, bacteria, phages,antibodies, antigens, DNA, RNA, receptors, enzymes, proteins,biochemicals, yeast, fungi, plant and animal cells and extracts, semen,sperm, ova, blood, tissue samples, cell samples, urine, saliva,lymphatic fluid, skin, hair, bones, or bone marrow.

In one embodiment, the biosensor may include a separate containerholding an effective amount of aqueous solution to restore thesuspension by irrigating the solid in ambient conditions with theaqueous solution. The aqueous solution used to irrigate the solid mayinclude distilled water, a buffer of 3-(N-morpholino) propanesulfonicacid, and one or more salts such as potassium chloride, sodium chloride,magnesium chloride, and/or calcium chloride.

In another aspect of the present invention, a method of reversiblypreserving a biological receptor includes the steps of combining thereceptor in its prepared condition with an effective amount of an AcaciaGum solution to form a suspension and, then, curing the suspension inambient conditions to form a solid. The preservation method may alsoinclude the steps of irrigating the solid in ambient conditions with aneffective amount of an aqueous solution to restore the suspension andthen separating the solution from the receptor to restore the receptorto its former, prepared condition.

In one embodiment, the Acacia Gum solution is formed by dissolving solidAcacia Gum in distilled water. The curing step may include stirring thesuspension.

In one embodiment, the aqueous solution used to irrigate the solid mayinclude distilled water, a buffer, and one or more salts such aspotassium chloride, sodium chloride, magnesium chloride, and/or calciumchloride. The buffer may be 3-(N-morpholino) propanesulfonic acid.

In another aspect of the invention, a method of fabricating a reversiblypreserved biological receptor disposed upon the interface of a biosensorincludes the steps of combining the biological receptor in its preparedcondition with an effective amount of an Acacia Gum solution to form asuspension and, then, curing the suspension in ambient conditions toform a solid that can later be restored to a suspension. In one aspect,the suspension is capable of being separated so that the biologicalreceptor can be restored to its former, prepared condition.

In one embodiment, the Acacia Gum solution used in this method offabrication is formed by dissolving solid Acacia Gum in distilled water.The curing step may include stirring the suspension. The combining stepmay include immersing the receptor.

In one embodiment, the method may include providing an effective amountof aqueous solution to restore the suspension by irrigating the solid inambient conditions with the aqueous solution. The aqueous solution usedto irrigate the solid may include distilled water, a buffer of3-(N-morpholino) propanesulfonic acid, and one or more salts such aspotassium chloride, sodium chloride, magnesium chloride, and/or calciumchloride.

In another aspect of the invention, a method of restoring the biologicalreceptor includes the steps of irrigating the solid in ambientconditions with an effective amount of an aqueous solution to restorethe suspension and, then, separating the solution from the biologicalreceptor such that the biological receptor is substantially restored toits former, prepared condition. In one embodiment, the aqueous solutionused to irrigate the solid may include distilled water, a buffer, andone or more salts such as potassium chloride, sodium chloride, magnesiumchloride, and/or calcium chloride. The buffer may be 3-(N-morpholino)propanesulfonic acid.

In another aspect of the invention, a water-soluble solid for reversiblypreserving a biological specimen includes a suspension formed bycombining the biological specimen in an isolated condition and aneffective amount of a solution of solid Acacia Gum dissolved in waterand an effective amount of aqueous solution to restore the suspension byirrigating the solid in ambient conditions with the aqueous solution.

In one embodiment, the aqueous solution used to irrigate the solid mayinclude distilled water, a buffer, and one or more salts such aspotassium chloride, sodium chloride, magnesium chloride, and/or calciumchloride. The buffer may be 3-(N-morpholino) propanesulfonic acid.

Thus, it is an object of the present invention to provide compositionsand methods for protecting and preserving biological samples withoutaltering or destroying the biological tissue. It is a related object toprovide preservation techniques that maintain the integrity and qualityof the biological sample.

It is a further object of the present invention to provide biologicalsamples that can be restored to their isolated or prepared state afterimmobilization, with minimal damage, for later study or use. It is arelated object of the present invention to provide a preservationtechnique that is both harmless and reversible.

It is a further object of the present invention to provide methods forrestoring biological specimens and receptors to their former conditionswithout a significant loss in viability or function.

It is another object of the present invention to provide biosensors withbiological receptors that can be restored to their prepared state afterimmobilization, with minimal damage, for later study or use.

It is yet another object of the present invention to provide awater-soluble solid for preserving biological specimens such that thespecimens can later be restored to their isolated state with minimaldamage.

These and other objects are accomplished by the method disclosed andwill become apparent from the following detailed description of onepreferred embodiment in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a photograph of Acacia Gum in powder form, liquid solution,and solid form.

FIG. 2 is a series of photographs of bacteria at various stages ofimmobilization and restoration, according to an embodiment of thepresent invention.

FIG. 3 is a photograph of crystal biosensors coated with a film ofAcacia Gum solution, according to an embodiment of the presentinvention.

FIG. 4 is a series of graphs representing the results of experimentationconducted according to an embodiment of the present invention.

DETAILED DESCRIPTION

The present invention, generally described, provides compositions andmethods for the preservation of biological samples. The compositionscomprise Acacia Gum, including derivations and modifications thereofwhich are useful as a reversible preservation solution. Acacia Gum is acomplex and highly branched carbohydrate polymer. The central core ornucleus is D-galactose and D-glucuronic acid, to which are attachedsugars such as L-arabinose, L-rhamnose, and the like. Acacia Gum isavailable as thin flakes, powder, granules, or angular fragments whichare completely soluble in hot and cold water.

Acacia Gum is a natural exudate or sap obtained from any of severalplants belonging to the genus Acacia. Acacia Senegal and Acacia Seyaltrees are the most commercially exploited species. Acacia Gum typicallyrefers to the gum harvested from Acacia Senegal trees. Acacia plants areleguminous shrubs and trees that grow in warm regions, such as theRepublic of the Sudan and the Upper Nile region of eastern Africa, wheremost of the world's Acacia Gum is harvested.

Acacia Gum was widely used in ancient Egypt in the preparation of inksand dyes and is thought to have been used as an adhesive formummification bindings. An article of commerce for centuries, the name“Arabic Gum” is believed to have been derived from the fact that Acaciagum was typically shipped from Arabian ports to Europe. Today, AcaciaGum is used in the manufacture of printing inks, textile dyes,adhesives, pharmaceuticals, vitamins, confections, foods, beverages,cosmetics, and many other products. For example, Acacia Gum is used tomake the water-soluble glue on postage stamps and envelopes, added tocandies to prevent crystallization, used as a coating to flavorparticles and beverages, added to beer to stabilize the foam, used as anemulsifier of fats in foods, lotions, and soaps, and is the mostimportant gum in the manufacture of ink.

The botanical name for the Acacia Gum referred to in this application isAcacia Nilotica (Linn.), N.O. Leguminosae. Acacia Gum is water-soluble,edible, non-toxic, highly uniform, pale in color, and has excellentemulsifying and film-forming qualities. Acacia Gum consists mainly ofhigh-molecular weight polysaccharides and their calcium, magnesium andpotassium salts.

Acacia Gum is harvested by tapping the trunk of an Acacia Senegal tree,which causes the gum to seep out and solidify into colorless or paleyellow tear-shaped nodules. The dried nodules are typically gathered byhand. Acacia Gum is commercially available in the form of white oryellowish flakes, granules, or powder. Acacia Gum powder is plentifuland readily available commercially, at a low cost. When the powder formis dissolved in water, the resulting solution becomes increasinglyviscous as the water evaporates, becoming a solid at room temperature.The photograph in FIG. 1 shows Acacia Gum powder in the vial on theleft, Acacia Gum in aqueous solution in the other vial, and between thevials a solid sheet of Acacia Gum at room temperature.

The compositions of the invention are useful for the preservation of anybiological sample of interest. Such samples include, without limitation,microorganisms, viruses, bacteria (such as as E. coli, Salmonella,Listeria, Staphylococcus, and others), phages, antibodies, antigens,DNA, RNA, receptors, enzymes, proteins, biochemicals, yeast and otherfungi, and plant and animal cells and extracts. Animal cells andextracts include, without limitation, semen, sperm, ova, blood, tissuesamples, cell samples, urine, saliva, lymphatic fluid, skin, hair,bones, and bone marrow. Additionally, biological samples includeproteins, enzymes, antibodies, monoclonal antibodies and the like.

The phrase, “biological specimen in an isolated condition,” as usedherein indicates a biological sample that has been isolated andsubstantially purified; meaning that it is substantially or essentiallyfree from components that normally accompany or interact with the sampleas found in its natural environment.

Isolation and Preservation Technique

Acacia Gum powder is readily soluble in water. The solution becomesincreasingly viscous as some of the water evaporates. An aqueous AcaciaGum solution is characterized by its reversibility. If more water isadded, the viscosity decreases. Even if the solution is permitted toharden or cure into a solid, the addition of water will return the solidto an aqueous solution. Reversibility in this context also refers to thefact that the Acacia Gum solution can be separated nearly completelyfrom the biological specimen after the preservation method of thepresent invention has been performed.

In one embodiment of the present invention, a biological specimen ispreserved by being immersed in or otherwise combined with an effectiveamount of Acacia Gum or an Acacia Gum solution. The amount of Acacia Gumsolution will vary depending upon sample size. The phrase “effectiveamount” is intended to indicate an amount sufficient to form asuspension; that is, to suspend the biological molecules or units of thespecimen within the Acacia Gum solution.

Initially upon being immersed in the solution, biological material suchas bacteria remain active and motile. As the viscosity increases,activity and motility decrease. In one embodiment, the suspension may bestirred to ensure a good distribution of specimen or to speed theevaporation of water and thus accelerate the curing process. Curing maytake place in ambient conditions; in other words, at room temperatureand at normal atmospheric pressures. When the solution solidifies, thebacteria shrink to about one-half to one-third of their original size.While the invention is not bound by any particular mechanism of action,it is postulated that the Acacia Gum solution penetrates the cellmembrane of the biological material, possibly replacing the water andresulting in the overall shrinkage observed. Inside the resulting solid,the bacteria remain dormant and may be kept at room temperature.

In one embodiment, the solid material containing the biological specimenmay be made into a powder, pellets, tablets, flakes, plates, capsules,or other forms or containers. The solid is transparent to visible light,a feature that makes it suitable for viewing and for certain opticalapplications. Moreover, although the solid is water-soluble, the solidis resistant to almost all organic solvents and most acids.

To restore the biological material to its isolated condition, the solidis irrigated with an aqueous solution. The amount of aqueous solutionneeded to change the solid back into a suspension will vary dependingupon the sample size. The phrase “effective amount of aqueous solution”is intended to indicate an amount sufficient to transform the solid intoa suspension.

In one aspect of the invention, the aqueous solution used to irrigatethe solid contains distilled water, a buffer, and one or more saltcompounds such as potassium chloride, sodium chloride, magnesiumchloride, and calcium chloride. The buffer is a substance capable insolution of neutralizing both acids and bases and, thereby, maintainingthe original pH of the solution. One such pH buffer in common use is3-(N-morpholino) propanesulfonic acid (also known as MOPS). Anothercommon pH buffer is called a phosphate buffer. A phosphate buffer, inone form, contains anhydrous monosodium phosphate and trisodiumphosphate dodecahydrate. A phosphate buffer solution may containdifferent molar ratios of monosodium phosphate and trisodium phosphate,depending upon the value of the pH to be maintained.

When irrigated, the solid gradually dissolves and the biologicalspecimen is again suspended within an Acacia Gum solution. The viscosityof the suspension decreases as more aqueous solution is added. Thebiological specimen returns to its normal size, absorbing the water lostor exchanged during the curing process.

In another aspect of the present invention, the suspension of biologicalmaterial and Acacia Gum solution is reversible because it can beseparated. The Acacia Gum solution can be removed using common methodsof separating mixtures, leaving the biological specimen in its isolatedcondition. The separation step restores the biological specimen to itsformer isolated or prepared condition. The phrase “substantiallyrestored” is intended to describe the nearly complete separation of theAcacia Gum solution from the biological specimen and the nearly completerestoration of viability of the biological specimen.

Biosensors

The methods of the invention find particular use in preservingbiological samples on biosensors. A biosensor, as shown in FIG. 3, iscomprised of a biological receptor, an interface, and a signaltransducer. The biochemical signal produced when a sample is placed onthe biological receptor is converted or translated by the signaltransducer into a quantifiable electrical signal.

The biological receptor is selected to sense a specific target compoundcalled the analyte. For example, a copper receptor will absorb coppermolecules from a sample. The signal transducer converts the activity onthe receptor (e.g., the accumulation of copper molecules) into anelectrical signal. For example, the signal transducer can detect theincreased mass of the biosensor by sensing changes in certain electricalproperties.

The types of biological receptors in use include, without limitation,enzymes, antibodies, phages, and lipid layers. The biological receptormust be prepared such that it will respond to the analyte. Preparationof the biological receptor includes depositing the biological materialonto the interface. Preparation of the interface to receive thebiological receptor may include chemical etching of the interface, theapplication of thin membranes, coating the interface with a thin layerof a particular biochemical to serve as an anchor for the biologicalreceptor, or any other of a variety of preparation methods. The phrase,“biological specimen in a prepared condition,” as used herein indicatesa biological receptor that has been isolated and deposited upon thebiosensor interface using any preparation technique that renders thereceptor ready for its intended use.

The signal transducer is typically an electrode connected to theinterface to measure any change in the receptor when the sample isintroduced. Signal transducer systems include, without limitation,piezoelectric crystals, conductimeters, enzyme-sensing electrodes,thermistors, optoelectronic and fiber-optic devices, field-effecttransistors, gas-sensing electrodes, and ion-selective electrodes. Thesignal transducer itself may be a pH-electrode, an oxygen electrode, ora piezoelectric crystal.

In a common biosensor using quartz crystal technology, shown in FIG. 3,the biological receptor is deposited in a film onto a piezoelectriccrystal, which serves as the interface. An electrode attached to thecrystal acts as the signal transducer. The quartz crystal is oscillatedat a known frequency based on its total mass, including the mass of thefilm receptor. When a sample containing the analyte is placed on thereceptor, the total mass will change when the antibodies in the receptorbind to the analyte. In response to the change in mass, the frequency ofthe crystal oscillation will change, and the change in frequency ismeasured by the signal transducer. Because frequency and mass arerelated, the additional mass can be calculated, indicating the preciseamount of the analyte present in the sample.

The Biosensor Experiment

A biosensor with a biological receptor comprised of antibodies againstSalmonella bacteria was covered with a film of Acacia Gum solution.After curing and storage at room temperature for a period of four (4)days, the antibodies were released by irrigation with water containing55.0 milli-Molar potassium chloride, 4.0 milli-Molar sodium chloride,1.0 milli-Molar magnesium chloride, 0.1 milli-Molar calcium chloride,and 2.0 milli-Molar 3-(N-morpholino) propanesulfonic acid, used as a pHbuffer. Preliminary data was obtained demonstrating the sensitivity ofthe restored sensors compared to the uncoated sensors, as shown in FIG.4 and Table One. TABLE ONE Performance of Coated Salmonella Biosensors.Coated Coated Uncoated (Group 1) (Group 2) Total Sensors 9 4 22 GoodSensors 4 1 8 Yield (%) 44.4% 25.0% 36.4% Slope (mV per decade) 15.3 7.619.4

Measurements were carried out with a Quartz Crystal Microbalance (QCM)measurement system. More specifically, the biosensors used in thisexperiment were the PM-700 series quartz sensor crystals available fromMaxtek, Inc. The output of the sensor crystal corresponds to the changein total mass. The signal transducer measures the change in the crystalin millivolts (mV). Referring to Table One and the graphs shown in FIG.4, the “mV per decade” refers to the voltage change for each order ofmagnitude change in the bacterial concentration.

The bacterial suspension of approximately 10⁹ cells per milliliter wasdiluted 10, 100, and 1000 times, respectively. The relativeconcentrations of bacteria were, therefore, 1, 10⁻¹, 10⁻², and 10⁻³.Accordingly, the logarithms (shown in FIG. 4) of the relativeconcentrations were 0, −1, −2, and −3, respectively.

For purposes of this experiment, a “good sensor” has a sensitivity ofmore than 7.0 mV per decade. The observation that only 44.4 percent ofthe uncoated biosensors were “good sensors” indicates the inherentfragility of the biological receptors used in biosensors.

The slope of the graphs shown in FIG. 4 indicates the degree ofsensitivity of the biosensor. The uncoated biosensors had a sensitivityof 15.3 mV per decade. While the sensitivity of Group 1 decreased to 7.6mV, the sensitivity of the coated biosensors in Group 2 was observed tobe 19.4 mV—better than the sensitivity of the uncoated sensors. In bothcases, the biosensors which had been coated with the Acacia Gum solutionwere fully operational and ready to use.

The Bull Sperm Experiment

In another aspect, the methods of the invention are useful in preservinganimal cells and extracts, such as sperm. In another experiment, theisolation and preservation technique of the present invention was usedto temporarily and reversible preserve bull sperm.

A sample of bull sperm was immobilized in Acacia Gum solution, where itremained at room temperature for a period of four (4) days before beingreleased by irrigation with water. Although reproduction was not tested,the bull sperm showed no difference in motility when compared to theinitial sample.

The present invention may be used to preserve bull sperm for transportor storage, at room temperature, without significant damage to thesperm. The cryogenic preparation and storage of bull sperm is expensiveand destructive because of crystalline structures formed duringfreezing. In contrast, the present invention does not introduce crystalsor other destructive structures into the sample and it is much lessexpensive.

Bacterial Cultures

The methods of the present invention are also useful in preservingsamples of bacteria. Two separate experiments were conducted to test theresponse and subsequent viability of bacteria suspended within an AcaciaGum solution.

In a first experiment, separate samples of Escherichia coli 0157 (E.coli) bacteria and Salmonella bacteria were immobilized in Acacia Gumsolution, where each sample remained at room temperature for a period ofseven (7) days. The bacteria were released by irrigation with watercontaining a phosphate buffer (pH 7.4) containing 2.7 milli-Molarpotassium chloride and 137 milli-Molar sodium chloride. The releasedbacteria showed no difference in motility when compared to the initialculture. The bacteria reproduced normally.

FIG. 2 shows the Salmonella bacteria at different stages of theexperiment.

Slide a shows the bacteria immersed in the Acacia Gum solution. Slide bshows the bacteria immobilized within the Acacia Gum solution, which hasbecome a solid at room temperature. Notice that the bacteria in Slide bare somewhat smaller.

After remaining immobilized for seven (7) days, the bacteria wereirrigated with an aqueous solution. The restoration process is shown inSlides c, d, e, and f.

Slide c shows the condition of the bacteria after one minute. Somemotion was observed after two minutes, shown in Slide d. Slide e showsthe condition of the bacteria after three minutes. After ten minutes, asshown in Slide f, the bacteria have returned to their normal size,absorbing the water lost during the immobilization or curing process.

In a second experiment, two additional samples of E. coli and Salmonellabacteria were immobilized in Acacia Gum solution for a period oftwenty-one (21) days, with the same results. The bacteria showed nodifference in motility when compared to the initial culture and thebacteria reproduced normally.

Other Uses

The present invention offers a method of reversibly preservingbiological specimens in a variety of contexts. The isolation andpreservation techniques of the present invention could be used, withoutlimitation, for isolating microbial cultures for shipment, bloodisolation and storage, time-release capsules for pharmaceuticals,biodegradable packaging, soluble prostheses and implants, surgery, andforensics.

The Acacia Gum solution and the isolation and preservation techniques ofthe present invention represent a simple, rapid, and inexpensivealternative to many of the biological preservation techniques in usetoday. Acacia Gum is organic, water-soluble, bio-compatible,biodegradable, and non-toxic. The preservation of biological specimenswith Acacia Gum is reversible and causes little or no damage to thespecimen.

While this invention has been described in specific detail withreference to the disclosed embodiments, it will be understood that manyvariations and modifications may be effected without departing from theinvention as described in the appended claims.

1. A biosensor, comprising: a signal transducer; an interface connectedto said signal transducer; and a biological receptor combined with aneffective amount of an Acacia Gum solution to form a suspension, saidsuspension cured in ambient conditions to form a solid disposed uponsaid interface, such that said biological receptor is reversiblypreserved, and such that said biosensor may be stored in ambientconditions.
 2. The biosensor of claim 1, wherein said solid may bedissolved by irrigating with an aqueous solution, such that saidbiological receptor is substantially restored and said biosensor isready to use.
 3. The biosensor of claim 1, wherein said biologicalreceptor is selected from the group consisting of microorganisms,enzymes, antibodies, phages, proteins, and lipid layers.
 4. Thebiosensor of claim 1, wherein said Acacia Gum solution comprises aquantity of solid Acacia Gum dissolved in a quantity of distilled water.5. The biosensor of claim 1, wherein said aqueous solution comprises aquantity of distilled water, a buffer, and a chloride compound.
 6. Thebiosensor of claim 5, wherein said buffer comprises a quantity of3-(N-morpholino) propanesulfonic acid, and wherein said chloridecompound is selected from the group consisting of potassium chloride,sodium chloride, magnesium chloride, and calcium chloride.
 7. A methodof preparing a biosensor, comprising: combining a biological receptor ina prepared condition with an effective amount of an Acacia Gum solutionto form a suspension; curing said suspension in ambient conditions toform a solid disposed upon a biosensor interface, such that saidbiological receptor is reversibly preserved within said solid; andstoring said biosensor in ambient conditions.
 8. The method of claim 7,further comprising: irrigating said solid with an effective amount of anaqueous solution to restore said suspension; and separating saidsuspension such that said biological receptor is substantially restoredto said prepared condition and said biosensor is ready to use.
 9. Themethod of claim 7, further comprising: selecting said biologicalreceptor from the group consisting of microorganisms, enzymes,antibodies, phages, proteins, and lipid layers.
 10. The method of claim7, wherein said step of combining further comprises: dissolving aquantity of solid Acacia Gum in distilled water to form said Acacia Gumsolution.
 11. The method of claim 8, wherein said step of irrigatingfurther comprises: combining distilled water, a buffer, and a chloridecompound to form said aqueous solution.
 12. The method of claim 11,further comprising: selecting 3-(N-morpholino) propanesulfonic acid assaid buffer; and selecting said chloride compound from the groupconsisting of potassium chloride, sodium chloride, magnesium chloride,and calcium chloride.
 13. A biosensor, comprising: a signal transducer;an interface connected to said signal transducer; and a biologicalreceptor combined with an effective amount of an Acacia Gum solution toform a suspension, said suspension cured in ambient conditions to form asolid disposed upon said interface, such that said biological receptoris reversibly preserved, and such that said biosensor may be stored inambient conditions, and wherein said solid may be dissolved byirrigating with an aqueous solution, such that said biological receptoris substantially restored and said biosensor is ready to use.
 14. Thebiosensor of claim 13, wherein said biological receptor is selected fromthe group consisting of microorganisms, enzymes, antibodies, phages,proteins, and lipid layers.
 15. The biosensor of claim 13, wherein saidAcacia Gum solution comprises a quantity of solid Acacia Gum dissolvedin a quantity of distilled water.
 16. The biosensor of claim 13, whereinsaid aqueous solution comprises a quantity of distilled water, a buffer,and a chloride compound.
 17. The biosensor of claim 16, wherein saidbuffer comprises a quantity of 3-(N-morpholino) propanesulfonic acid,and wherein said chloride compound is selected from the group consistingof potassium chloride, sodium chloride, magnesium chloride, and calciumchloride.
 18. A method of preparing a biosensor, comprising: combining abiological receptor in a prepared condition with an effective amount ofan Acacia Gum solution to form a suspension; curing said suspension inambient conditions to form a solid disposed upon a biosensor interface,such that said biological receptor is reversibly preserved within saidsolid; storing said biosensor in ambient conditions; irrigating saidsolid with an effective amount of an aqueous solution to restore saidsuspension; and separating said suspension such that said biologicalreceptor is substantially restored to said prepared condition and saidbiosensor is ready to use.
 19. The method of claim 18, furthercomprising: selecting said biological receptor from the group consistingof microorganisms, enzymes, antibodies, phages, proteins, and lipidlayers.
 20. The method of claim 18, wherein said step of combiningfurther comprises: dissolving a quantity of solid Acacia Gum indistilled water to form said Acacia Gum solution.
 21. The method ofclaim 18, wherein said step of irrigating further comprises: combiningdistilled water, a buffer, and a chloride compound to form said aqueoussolution.
 22. The method of claim 21, further comprising: selecting3-(N-morpholino) propanesulfonic acid as said buffer; and selecting saidchloride compound from the group consisting of potassium chloride,sodium chloride, magnesium chloride, and calcium chloride.